Urine gene expression ratios for detection of cancer

ABSTRACT

This invention relates to methods for determining the presence of cancer in a subject based on the analysis of the expression levels of an under-expressed tumor marker (TM) and at least one other TM. Specifically, this invention relates to the determination of a cancer, particularly bladder cancer, by performing ratio, regression or classification analysis of the expression levels of at least one under-expressed TM, particularly an under-expressed bladder TM (BTM), and at least one over-expressed TM, particularly an over-expressed BTM. In various aspects, the invention relates to kits and devices for carrying out these methods.

CLAIM OF PRIORITY

This application is a Continuation application filed under 35 U.S.C.§111(a) and 37 C.F.R. §1.53(b) of non-provisional U.S. application Ser.No. 12/221,626, filed 5 Aug. 2008, which is a Continuation applicationfiled under 35 U.S.C. 111(a) of PCT International Application NumberPCT/NZ2007/000029, International Filing Date Feb. 9, 2007, InventorParry Guilford, entitled “Urine Gene Expression Ratios for Detection ofCancer.” The above PCT Application claims priority to New Zealandprovisional patent application number NZ 545,243, filed 10 Feb. 2006.Each of the above applications is expressly incorporated herein fully byreference, as if individually so incorporated.

TECHNICAL FIELD

This invention relates to detection of cancer. Specifically, theinvention relates to the use of markers for the detection of bladdercancer. More specifically, this invention relates to use of anunder-expressed marker in combination with at least one other marker forthe detection of bladder cancer.

BACKGROUND

Survival of cancer patients is greatly enhanced when the cancer istreated early. In the case of bladder cancer, patients diagnosed withearly stage disease have 5-year survival rates of >90%, compared toapproximately 15-30% for patients diagnosed with advanced disease.Therefore, developments that lead to early diagnosis of bladder cancercan lead to an improved prognosis for the patients. The establishedmethod for detecting bladder cancer using urine samples is cytology.However, cytology is known to be only about 75% sensitive for detectinginvasive bladder cancer and only about 25% sensitive for detectingsuperficial bladder cancer (Lotan and Roehrborn, Urology 61, 109-118(2003)).

Identification of specific markers for cancer in urine can provide avaluable approach for the early diagnosis of cancer, leading to earlytreatment and improved prognosis. Specific cancer markers also provide ameans for monitoring disease progression, enabling the efficacy ofsurgical, radiotherapeutic and chemotherapeutic treatments to bemonitored.

At present, the most reliable method for detecting bladder cancer iscystoscopy accompanied by histology of biopsied lesions. However, thistechnique is time consuming, invasive and its sensitivity is onlyapproximately 90%, meaning that about 10 percent of cancers are notdetected using these methods. Of the non-invasive methodologies, urinecytology, which detects exfoliated malignant cells microscopically, isthe current preferred method. Although cytology has a specificity ofabout 95%, it has poor sensitivity (9-25%) for low-grade lesions, isextremely dependent on sample quality and suffers from highinter-observer variability.

Several urine protein markers are known. Tests for these markers offerbetter sensitivity than cytology, but tend to suffer from sub-optimalspecificity because elevated levels of these markers are also commonlyobserved in patients with non-malignant diseases including inflammation,urolithiasis and benign prostatic hyperplasia. For example, NMP22, whichdetects a specific nuclear matrix protein, has a sensitivity of 47-87%and a specificity of 58-91%.

One drawback associated with urine testing is that individual markerlevels can vary significantly with: (i) different urine collectionmethods (catheterised, voided, urine pellets); (ii) the diurnal timingof urine sampling; (iii) the point of sampling during voiding (e.g.midstream vs end sample); and (iv) urine concentration associated withvarying fluid intake, kidney function or diseases that affect plasmavolume. These variations have the potential to lead to false positiveand false negative tests. Although some of this variation can be reducedusing strict standard operating procedures, patient compliance withthese procedures can be unreliable. The effect of varying urineconcentration can, in some instances, be accounted for by assessingmarker levels relative to urinary creatinine, however, this increasesthe cost and complexity of testing, particularly when sample preparationor storage methods differ for marker detection and creatininemeasurement.

There is a need for simple tools for the early detection and diagnosisof cancer. This invention provides further methods, devices and kitsbased on markers, specifically ratios, regression or classificationanalysis of bladder cancer markers, to aid in the detection anddiagnosis of bladder cancer.

SUMMARY OF THE INVENTION

The present invention provides for a method for determining the presenceof a cancer in a subject, comprising:

(a) providing a sample from the subject;

(b) detecting the expression level of at least two tumour marker (TM)family members in said sample, wherein at least one TM is anunder-expressed TM;

(c) establishing whether the patient has cancer according to apredetermined threshold.

Step (c) can be preformed by determining the ratio of expression of saidTMs, or by performing regression or classification analysis on the TMexpression levels.

The TM can be a BTM. The cancer to be detected can be bladder cancer,and in certain embodiments at least one of the TMs is an over-expressedBTM. The over-expressed BTM may be selected from the group outlined inFIG. 11 or FIG. 12.

In certain embodiments at least one under-expressed TM is a BTM selectedfrom the group outlined in FIG. 3 or FIG. 4.

In other embodiments of the present invention the step of detecting iscarried out by detecting over expression of BTM mRNA, a BTM protein, ora BTM peptide.

The sample can be any one of biopsy, blood, serum, peritoneal washes,cerebrospinal fluid, urine and stool samples

The present invention also provides for a device for detecting a TM,comprising:

a substrate having a TM capture reagent thereon; and

a detector associated with said substrate, said detector capable ofdetecting a TM associated with said capture reagent, wherein the TM isan under-expressed TM.

The TM can be a BTM.

The TM capture reagent can be an oligonucleotide or an antibody.

In certain embodiments the TM can be a BTM selected from the groupoutlined in FIG. 3 or FIG. 4.

The present invention also provides for a kit for determining thepresence of a cancer in a subject, comprising:

a substrate;

at least two TM capture reagents, wherein at least one TM is anunder-expressed TM; and

instructions for use.

The TM can be a BTM.

The TM capture reagent may be a TM-specific oligonucleotide or aTM-specific antibody.

The TM detected by the kit may be a BTM selected from the group outlinedin FIG. 3 or FIG. 4.

At least one of the TMs detected by the kit may be an over-expressed TMor an over-expressed BTM. The over-expressed BTM may be selected fromthe group outlined in FIG. 11 or FIG. 12.

BRIEF DESCRIPTION OF THE FIGURES

This invention is described with reference to specific embodimentsthereof and with reference to the Figures, in which:

FIG. 1 depicts a table showing the characteristics of urine samples usedin the qPCR analyses.

FIG. 2 depicts a table of primers and oligonucleotide probes of markersfor qPCR analysis of bladder cancer according to the present invention.

FIG. 3 depicts a table of under-expressing bladder tumour markersidentified using microarray methods on samples of bladder cancer.

FIG. 4 depicts a table of under-expressing bladder tumour markersidentified using microarray methods on samples of bladder cancer thathave insignificant expression in whole blood, but high expression innormal bladder tissue.

FIG. 5 depicts box and whisker plots showing the ratios of three bladdertransitional cell carcinoma (TCC) markers (HoxA13, IGFBP5, and MDK) withthe under expressing marker LTB4DH for urine samples from patients witheither non-malignant urological disease or TCC. The boxes define the25^(th), 50^(th) and 75^(th) percentiles and the horizontal bars markthe adjacent values. Outliers are shown by circles. The unfilled boxesrepresent samples from non-malignant disease controls and the shadedboxes represent samples from patients with TCC.

FIG. 6 shows examples of the sensitivities and specificities of TCCdetection for tests that include LTB4DH. (a). single tests; (b).combination tests using LTB4DH and two of the three markers HoxA13,IGFBP5, and MDK.

FIG. 7a-c shows ROC curves for the sensitivity and specificity ofdetection of TCC in urine samples using ratios that include LTB4DH. 7 a.IGFBP5/LTB4DH; 7 b. MDK/LT4BDH; 7 c. HoxA13/LTB4DH.

FIG. 8a-f shows scatter plots for combination tests, a-c using LTB4DHand two of the three markers HoxA13, IGFBP5, and MDK, and d-f repeatedusing BAG1 for LTB4DH. 8 a. MDK/LTB4DH and IGFBP5/LTB4DH; 8 b.MDK/LTB4DH and HoxA13/LTB4DH; IGFBP5/LTB4DH and HoxA13/LTB4DH; 8 dMDK/BAG1 and IGFBP5/BAG1; 8 e.MDK/BAG1 and HoxA13/BAG1; 8 f. IGFBP5/BAG1and HoxA13/BAG1.

FIG. 9a-b shows scatter plots showing the correlation between ΔCt forIGFBP5 and ΔCt ratios for IGFBP5/LTB4DH and urine creatinineconcentration. 9 a. Urine samples from patients with TCC 9 b. Urinesamples from patients with non-malignant disease

FIG. 10a-f depicts self-self scatter plots showing the distribution ofvoided and catheterised urine samples from TCC patients analysed usingthe bladder tumour markers MDK, IGFBP5 and HoxA13 alone or in ratioswith LTB4DH.

FIG. 11 shows known over-expressed markers from invasive bladdertumours.

FIG. 12 shows known over-expressed markers from superficial bladdertumours.

FIG. 13 shows the clinical characteristics of low grade TCC samples andcontrols used in ROC curve analysis.

FIG. 14 shows the results of a ROC Curve analysis. Illustration of theincreased test accuracy obtained when LTB4DH is used in ratios withHoxA13 and IGFBP5.

FIG. 15 shows the results of a Linear Discriminate Analysis of BTMs,with and without LTB4DH, for the detection of TCC.

DETAILED DESCRIPTION

Definitions

The term “marker” means a molecule that is associated quantitatively orqualitatively with the presence of a biological phenomenon. Examples of“markers” include a polynucleotide, such as a gene, gene fragment, RNA,or RNA fragment; or a gene product, including a polypeptide such as apeptide, oligopeptide protein or protein fragment; or relatedmetabolites, by products or other identifying molecules, such asantibodies or antibody fragments whether related directly or indirectlyto a mechanism underlying the phenomenon. The markers of the inventioninclude the nucleotide sequences (e.g. GenBank sequences) as disclosedherein, in particular the full length sequences, any coding sequences,non-coding sequences and fragments, or any compliments thereof, and anymeasurable marker thereof as defined above.

The term “sensitivity” means the proportion of individuals with thedisease who test (by the model) positive. Thus, increased sensitivitymeans fewer false negative test results.

The term “specificity” means the proportion of individuals without thedisease who test (by the model) negative. Thus, increased specificitymeans fewer false positive test results. The term “expression” includesproduction of polynucleotides and polypeptides, in particular, theproduction of RNA (e.g., mRNA) from a gene or portion of a gene, andincludes the production of a polypeptide encoded by an RNA or gene orportion of a gene, and includes appearance of a detectable materialassociated with expression. For example, the formation of a complex, forexample, from a polypeptide-polypeptide interaction,polypeptide-nucleotide interaction, or the like, is included within thescope of the term “expression”. Another example, the binding of abinding ligand, such as a hybridization probe or antibody, to a gene orother polynucleotide, a polypeptide or a protein fragment and thevisualization of the binding ligand Thus, the density of a spot on amicroarray, on a hybridization blot such as a Northern blot, or on animmunoblot, such as a Western blot, or on a bead array, or by PCRanalysis, is included within the term “expression” of the underlyingbiological molecule.

The term “over expression” is used where the expression of a marker inone cell, or cell type, is greater than that of another equivalent cell,or cell type.

The term “under expression” is used where the expression of a marker inone cell, or cell type, is less than that of another equivalent cell, orcell type.

The term “TM” or “tumour marker” or “TM family member” means a markerthat is associated with a particular cancer. The term TM also includescombinations of individual markers, whose combination improves thesensitivity and specificity of detecting cancer. It is to be understoodthat the term TM does not require that the marker be specific only for aparticular tumour. Rather, expression of TM can be altered in othertypes of cells, diseased cells, tumours, including malignant tumours.

A TM can be identified by extracting RNA from a tissue sample from apatient suspected of having bladder cancer, applying the RNA or cDNAcopy to a microarray having a number of oligonucleotides thereon,permitting the sample RNA to hybridize to the oligonucleotides on thearray, and then quantifying the level of measured RNA bound to the eacharray spot. A marker is considered to be a under expressing TM if itspresence is below a threshold of at least about 1.2 times that found innormal, non-malignant tissue using microarray methods. Alternatively,the threshold can be below about 2 times normal, about 3 times less thannormal, 4 times or even about 5 times less than normal. By “normal” wemean less than the 90^(th) percentile of the normal population. In othercases, normal can mean a level of presence of the 95^(th) percentile(i.e., about 2 Standard Deviations (SD) from the mean), and in othercases, less than about 97.5^(th) percentile (i.e., about 3 SD) or the99^(th) percentile.

The term “under expressing TM” means a marker that shows lowerexpression in bladder tumours than in non-malignant bladder tissue. Theterm “over expressing TM” means a marker that shows higher expression inbladder tumours than in non-malignant tissue.

The term “BTM” or “bladder tumour marker” or “BTM family member” means aTM that is associated with bladder cancer. The term BTM also includescombinations of individual markers, whose combination improves thesensitivity and specificity of detecting bladder cancer. It is to beunderstood that the term BTM does not require that the marker bespecific only for bladder tumours. Rather, expression of BTM can bealtered in other types of cells, diseased cells, tumours, includingmalignant tumours.

The term “under expressing BTM” means a marker that shows lowerexpression in bladder tumours than in non-malignant bladder tissue.

The term “over expressing BTM” means a marker that shows higherexpression in bladder tumours than in non-malignant tissue.

The term “qPCR” means quantitative polymerase chain reaction. The term“qPCR” or “QPCR” refers to quantative polymerase chain reaction asdescribed, for example, in PCR Technique: Quantitative PCR, J. W.Larrick, ed., Eaton Publishing, 1997, and A-Z of Quantitative PCR, S.Bustin, ed., IUL Press, 2004.

The term “TCC” means transitional cell carcinoma of the bladder. TCCsconstitute ˜95% of all bladder cancers.

As used herein “antibodies” and like terms refer to immunoglobulinmolecules and immunologically active portions of immunoglobulin (Ig)molecules, i.e., molecules that contain an antigen binding site thatspecifically binds (immunoreacts with) an antigen. These include, butare not limited to, polyclonal, monoclonal, chimeric, single chain, Fc,Fab, Fab′, and Fab2 fragments, and a Fab expression library. Antibodymolecules relate to any of the classes IgG, IgM, IgA, IgE, and IgD,which differ from one another by the nature of heavy chain present inthe molecule. These include subclasses as well, such as IgG1, IgG2, andothers. The light chain may be a kappa chain or a lambda chain.Reference herein to antibodies includes a reference to all classes,subclasses, and types. Also included are chimeric antibodies, forexample, monoclonal antibodies or fragments thereof that are specific tomore than one source, e.g., a mouse or human sequence. Further includedare camelid antibodies, shark antibodies or nanobodies.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byabnormal or unregulated cell growth. Cancer and cancer pathology can beassociated, for example, with metastasis, interference with the normalfunctioning of neighbouring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

The term “tumour” refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

The term “microarray” refers to an ordered or unordered arrangement ofcapture agents, preferably polynucleotides (e.g., probes) orpolypeptides on a substrate. See, e.g., Microarray Analysis, M. Schena,John Wiley & Sons, 2002; Microarray Biochip Technology, M. Schena, ed.,Eaton Publishing, 2000; Guide to Analysis of DNA Microarray Data, S.Knudsen, John Wiley & Sons, 2004; and Protein Microarray Technology, D.Kambhampati, ed., John Wiley & Sons, 2004.

The term “oligonucleotide” refers to a polynucleotide, typically a probeor primer, including, without limitation, single-strandeddeoxyribonucleotides, single- or double-stranded ribonucleotides,RNA:DNA hybrids, and double-stranded DNAs. Oligonucleotides, such assingle-stranded DNA probe oligonucleotides, are often synthesized bychemical methods, for example using automated oligonucleotidesynthesizers that are commercially available, or by a variety of othermethods, including in vitro expression systems, recombinant techniques,and expression in cells and organisms.

The term “polynucleotide,” when used in the singular or plural,generally refers to any polyribonucleotide or polydeoxribonucleotide,which may be unmodified RNA or DNA or modified RNA or DNA. Thisincludes, without limitation, single- and double-stranded DNA, DNAincluding single- and double-stranded regions, single- anddouble-stranded RNA, and RNA including single- and double-strandedregions, hybrid molecules comprising DNA and RNA that may besingle-stranded or, more typically, double-stranded or include single-and double-stranded regions. Also included are triple-stranded regionscomprising RNA or DNA or both RNA and DNA. Specifically included aremRNAs, cDNAs, and genomic DNAs, and any fragments thereof. The termincludes DNAs and RNAs that contain one or more modified bases, such astritiated bases, or unusual bases, such as inosine. The polynucleotidesof the invention can encompass coding or non-coding sequences, or senseor antisense sequences. It will be understood that each reference to a“polynucleotide” or like term, herein, will include the full-lengthsequences as well as any fragments, derivatives, or variants thereof.

“Polypeptide,” as used herein, refers to an oligopeptide, peptide, orprotein sequence, or fragment thereof, and to naturally occurring,recombinant, synthetic, or semi-synthetic molecules. Where “polypeptide”is recited herein to refer to an amino acid sequence of a naturallyoccurring protein molecule, “polypeptide” and like terms, are not meantto limit the amino acid sequence to the complete, native amino acidsequence for the full-length molecule. It will be understood that eachreference to a “polypeptide” or like term, herein, will include thefull-length sequence, as well as any fragments, derivatives, or variantsthereof.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridisable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. Additional details andexplanation of stringency of hybridization reactions, are found e.g., inAusubel et al., Current Protocols in Molecular Biology, WileyInterscience Publishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, typically: (1) employ low ionic strength and high temperaturefor washing, for example 0.015 M sodium chloride/0.0015 M sodiumcitrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ a denaturingagent during hybridization, such as formamide, for example, 50% (v/v)formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×, Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washcomprising 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e. g., temperature, ionic strength, and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, and biochemistry,which are within the skill of the art. Such techniques are explainedfully in the literature, such as, Molecular Cloning: A LaboratoryManual, 2nd edition, Sambrook et al., 1989; Oligonucleotide Synthesis, MJ Gait, ed., 1984; Animal Cell Culture, R. I. Freshney, ed., 1987;Methods in Enzymology, Academic Press, Inc.; Handbook of ExperimentalImmunology, 4th edition, D. M. Weir & C C. Blackwell, eds., BlackwellScience Inc., 1987; Gene Transfer Vectors for Mammalian, Cells, J. M.Miller & M. P. Calos, eds., 1987; Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., 1987; and PCR: The Polymerase ChainReaction, Mullis et al., eds., 1994.

Description of Embodiments of the Invention

Using a combination of microarray analysis and quantitative polymerasechain reaction (qPCR), markers for transitional cell carcinoma of thebladder (TCC) that are under-expressed in tumours have been identified.It has surprisingly been found that ratios between these markers andother bladder tumour markers (BTM), especially markers that are overexpressed in tumours, are diagnostic for bladder cancer.

The ratios (rather than measuring an absolute level of a marker)identifies a simple gene expression ‘signature’ that typifies bladdercancer cells, and surprisingly is more robust to variations in samplingtechniques or urine concentration. Moreover, the combination of anunder-expressed marker and an over-expressed marker maximizes thedifferential between samples from patients and non-malignant controls,increasing the test reliability. The under-expressed markers describedhere have been selected on the basis of (i) strong and consistentdown-regulation in TCC, (ii) high expression in normal tissue, and (iii)insignificant expression in whole blood to minimize the risk of falsepositives in patients presenting with hematuria.

As an alternative to determining the ration of the two BTM,s it has alsobeen found that the under-expressed and over-expressed BTMs can beanalysed in regression analyses or classification techniques includinglinear discriminate analysis, and the results of these analyses are alsoindicative of the presence of bladder cancer.

The test involves the measuring of at least two TM markers, such as aBTM, in a sample from a patient suspected of having a cancer or at riskof having cancer, wherein at least one of the TMs is an under-expressedTM. The ratio of the under-expressed TM and the other TM is indicativeof the presence of cancer. The second TM can be any TM as known in theart, but preferably is an over-expressed BTM, FIG. 3 shows a number ofunder-expressed markers suitable for use in the present invention.

The test is best preformed using an under-expressed TM in combinationwith an over-expressed TM. Any over-expressed TM can be used, forexample. Known over expressed BTMs identified from invasive bladdertumours (defined here as tumours≧stage 1), are outlined in FIG. 11, andover-expressed BTMs identified from superficial bladder tumours (definedhere as Stage Ta and Tis tumours) are shown in FIG. 12.

It has also been surprisingly established that preferred under-expressedBTMs for use in the present invention are ones that are notsignificantly elevated in whole blood, and are present in sufficientlyhigh copy numbers in both tumour cells and non-malignant bladder cells.Preferred under-expressed BTMs are outlined in FIG. 4.

Cancer markers can be detected in a sample using any suitable technique,and can include, but are not limited to, oligonucleotide probes, qPCR orantibodies raised against cancer markers.

It will be appreciated that the sample to be tested is not restricted toa sample of the tissue suspected of being a tumour. The marker may besecreted into the serum, sloughed from cell membranes, released fromlysed cells or associated with cells lost into the urine. Therefore, asample can include any bodily sample, and includes biopsies, blood,serum, peritoneal washes, cerebrospinal fluid, urine and stool samples.

It will also be appreciate that the present invention is not restrictedto the detection of cancer in humans, but is suitable for the detectionof cancer in any animal, including, but not limited to dogs, cats,horses, cattle, sheep, deer, pigs and any other animal known to getcancer.

General Approaches to Cancer Detection

The following approaches are non-limiting methods that can be used tomeasure TMs. Following measurement of individual TMs, ratios betweenhigh and low expressing BTM family members are determined. These ratiosare used to predict the presence or absence cancer.

Alternatively, the high and low expressing TMs are used in regression orclassification analyses. The results of these analyses are also used topredict the presence or absence cancer.

General methodologies for determining expression levels are outlinedbelow, although it will be appreciated that any method for determiningexpression levels would be suitable.

Quantitative PCR (qPCR)

Quantitative PCR (qPCR) can be carried out on tumour samples, on serum,plasma and urine samples using BTM specific primers and probes. Incontrolled reactions, the amount of product formed in a PCR reaction(Sambrook, J., E Fritsch, E. and T Maniatis, Molecular Cloning: ALaboratory Manual 3^(rd). Cold Spring Harbor Laboratory Press: ColdSpring Harbor (2001)) correlates with the amount of starting template.Quantification of the PCR product can be carried out by stopping the PCRreaction when it is in log phase, before reagents become limiting. ThePCR products are then electrophoresed in agarose or polyacrylamide gels,stained with ethidium bromide or a comparable DNA stain, and theintensity of staining measured by densitometry. Alternatively, theprogression of a PCR reaction can be measured using PCR machines such asthe Applied Biosystems' Prism 7000 or the Roche LightCycler whichmeasure product accumulation in real-time. Real-time PCR measures eitherthe fluorescence of DNA intercalating dyes such as Sybr Green into thesynthesized PCR product, or the fluorescence released by a reportermolecule when cleaved from a quencher molecule; the reporter andquencher molecules are incorporated into an oligonucleotide probe whichhybridizes to the target DNA molecule following DNA strand extensionfrom the primer oligonucleotides. The oligonucleotide probe is displacedand degraded by the enzymatic action of the Taq polymerase in the nextPCR cycle, releasing the reporter from the quencher molecule. In onevariation, known as Scorpion®, the probe is covalently linked to theprimer.

Reverse Transcription PCR (RT-PCR)

RT-PCR can, be used to compare RNA levels in different samplepopulations, in normal and tumour tissues, with or without drugtreatment, to characterize patterns of expression, to discriminatebetween closely related RNAs, and to analyze RNA structure.

For RT-PCR, the first step is the isolation of RNA from a target sample.The starting material is typically total RNA isolated from human tumoursor tumour cell lines, and corresponding normal tissues or cell lines,respectively. RNA can be isolated from a variety of samples, such astumour samples from breast, lung, colon (e.g., large bowel or smallbowel), colorectal, gastric, esophageal, anal, rectal, prostate, brain,liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, bladderetc., tissues, from primary tumours, or tumour cell lines, and frompooled samples from healthy donors. If the source of RNA is a tumour,RNA can be extracted, for example, from frozen or archivedparaffin-embedded and fixed (e.g., formalin-fixed) tissue samples.

The first step in gene expression profiling by RT-PCR is the reversetranscription of the RNA template into cDNA, followed by its exponentialamplification in a PCR reaction. The two most commonly used reversetranscriptases are avian myeloblastosis virus reverse transcriptase(AMV-RT) and Moloney murine leukaemia virus reverse transcriptase(MMLV-RT). The reverse transcription step is typically primed usingspecific primers, random hexamers, or oligo-dT primers, depending on thecircumstances and the goal of expression profiling. For example,extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit(Perkin Elmer, Calif., USA), following the manufacturer's instructions.The derived cDNA can then be used as a template in the subsequent PCRreaction.

Although the PCR step can use a variety of thermostable DNA-dependentDNA polymerases, it typically employs the Taq DNA polymerase, which hasa 5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonucleaseactivity. Thus, TaqMan (q) PCR typically utilizes the 5′ nucleaseactivity of Taq or Tth polymerase to hydrolyze a hybridization probebound to its target amplicon, but any enzyme with equivalent 5′ nucleaseactivity can be used.

Two oligonucleotide primers are used to generate an amplicon typical ofa PCR reaction. A third oligonucleotide, or probe, is designed to detectnucleotide sequence located between the two PCR primers. The probe isnon-extendible by Taq DNA polymerase enzyme, and is labeled with areporter fluorescent dye and a quencher fluorescent dye. Anylaser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

TaqMan RT-PCR can be performed using commercially available equipment,such as, for example, ABI PRISM 7700 Sequence Detection System(Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), orLightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In apreferred embodiment, the 5′ nuclease procedure is run on a real-timequantitative PCR device such as the ABI PRISM 7700tam Sequence DetectionSystem.

The system consists of a thermocycler, laser, charge-coupled device(CCD), camera, and computer. The system amplifies samples in a 96-wellformat on a thermocycler. During amplification, laser-inducedfluorescent signal is collected in real-time through fibre optics cablesfor all 96 wells, and detected at the CCD. The system includes softwarefor running the instrument and for analyzing the data.

5′ nuclease assay data are initially expressed as Ct, or the thresholdcycle. As discussed above, fluorescence values are recorded during everycycle and represent the amount of product amplified to that point in theamplification reaction. The point when the fluorescent signal is firstrecorded as statistically significant is the threshold cycle.

Real-Time Quantitative PCR (qPCR)

A more recent variation of the RT-PCR technique is the real timequantitative PCR, which measures PCR product accumulation through adual-labeled fluorigenic probe (i.e., TaqMan probe). Real time PCR iscompatible both with quantitative competitive PCR and with quantitativecomparative PCR. The former uses an internal competitor for each targetsequence for normalization, while the latter uses a normalization genecontained within the sample, or a housekeeping gene for RT-PCR. Furtherdetails are provided, e.g., by Held et al., Genome Research 6: 986-994(1996).

Expression levels can be determined using fixed, paraffin-embeddedtissues as the RNA source. According to one aspect of the presentinvention, PCR primers and probes are designed based upon intronsequences present in the gene to be amplified. In this embodiment, thefirst step in the primer/probe design is the delineation of intronsequences within the genes. This can be done by publicly availablesoftware, such as the DNA BLAT software developed by Kent, W. J., GenomeRes. 12 (4): 656-64 (2002), or by the BLAST software including itsvariations. Subsequent steps follow well established methods of PCRprimer and probe design.

In order to avoid non-specific signals, it is useful to mask repetitivesequences within the introns when designing the primers and probes. Thiscan be easily accomplished by using the Repeat Masker program availableon-line through the Baylor College of Medicine, which screens DNAsequences against a library of repetitive elements and returns a querysequence in which the repetitive elements are masked. The maskedsequences can then be used to design primer and probe sequences usingany commercially or otherwise publicly available primer/probe designpackages, such as Primer Express (Applied Biosystems); MGBassay-by-design (Applied Biosystems); Primer3 (Steve Rozen and Helen J.Skaletsky (2000) Primer3 on the WWW for general users and for biologistprogrammers in: Krawetz S, Misener S (eds) Bioinformatics Methods andProtocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp365-386).

The most important factors considered in PCR primer design includeprimer length, melting temperature (Tm), and G/C content, specificity,complementary primer sequences, and 3′ end sequence. In general, optimalPCR primers are generally 17-30 bases in length, and contain about20-80%, such as, for example, about 50-60% G+C bases. Meltingtemperatures between 50 and 80° C., e.g., about 50 to 70° C., aretypically preferred. For further guidelines for PCR primer and probedesign see, e.g., Dieffenbach, C. W. et al., General Concepts for PCRPrimer Design in: PCR Primer, A Laboratory Manual, Cold Spring HarborLaboratory Press, New York, 1995, pp. 133-155; Innis and Gelfand,Optimization of PCRs in: PCR Protocols, A Guide to Methods andApplications, CRC Press, London, 1994, pp. 5-11; and Plasterer, T. N.Primerselect: Primer and probe design. Methods Mol. Biol. 70: 520-527(1997), the entire disclosures of which are hereby expresslyincorporated by reference.

Microarray Analysis

Differential expression can also be identified, or confirmed using themicroarray technique. Thus, the expression profile of CCPMs can bemeasured in either fresh or paraffin-embedded tumour tissue, usingmicroarray technology. In this method, polynucleotide sequences ofinterest (including cDNAs and oligonucleotides) are plated, or arrayed,on a microchip substrate. The arrayed sequences (i.e., capture probes)are then hybridized with specific polynucleotides from cells or tissuesof interest (i.e., targets). Just as in the RT-PCR method, the source ofRNA typically is total RNA isolated from human tumours or tumour celllines, and corresponding normal tissues or cell lines. Thus RNA can beisolated from a variety of primary tumours or tumour cell lines. If thesource of RNA is a primary tumour, RNA can be extracted, for example,from frozen or archived formalin fixed paraffin-embedded (FFPE) tissuesamples and fixed (e.g., formalin-fixed) tissue samples, which areroutinely prepared and preserved in everyday clinical practice.

In a specific embodiment of the microarray technique, PCR amplifiedinserts of cDNA clones are applied to a substrate. The substrate caninclude up to 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 75nucleotide sequences. In other aspects, the substrate can include atleast 10,000 nucleotide sequences. The microarrayed sequences,immobilized on the microchip, are suitable for hybridization understringent conditions. As other embodiments, the targets for themicroarrays can be at least 50, 100, 200, 400, 500, 1000, or 2000 basesin length; or 50-100, 100-200, 100-500, 100-1000, 100-2000, or 500-5000bases in length. As further embodiments, the capture probes for themicroarrays can be at least 10, 15, 20, 25, 50, 75, 80, or 100 bases inlength; or 10-15, 10-20, 10-25, 10-50, 10-75, 10-80, or 20-80 bases inlength.

Fluorescently labeled cDNA probes may be generated through incorporationof fluorescent nucleotides by reverse transcription of RNA extractedfrom tissues of interest. Labeled cDNA probes applied to the chiphybridize with specificity to each spot of DNA on the array. Afterstringent washing to remove non-specifically bound probes, the chip isscanned by confocal laser microscopy or by another detection method,such as a CCD camera. Quantitation of hybridization of each arrayedelement allows for assessment of corresponding mRNA abundance. With dualcolour fluorescence, separately labeled cDNA probes generated from twosources of RNA are hybridized pairwise to the array. The relativeabundance of the transcripts from the two sources corresponding to eachspecified gene is thus determined simultaneously. An exemplary protocolfor this is described in detail in Example 4.

The miniaturized scale of the hybridization affords a convenient andrapid evaluation of the expression pattern for large numbers of genes.Such methods have been shown to have the sensitivity required to detectrare transcripts, which are expressed ata few copies per cell, and toreproducibly detect at least approximately two-fold differences in theexpression levels (Schena et al., Proc. Natl. Acad. Sci. USA 93 (2):106-149 (1996)). Microarray analysis can be performed by commerciallyavailable equipment, following manufacturer's protocols, such as byusing the Affyrnetrix GenChip technology, Illumina microarray technologyor Incyte's microarray technology. The development of microarray methodsfor large-scale analysis of gene expression makes it possible to searchsystematically for molecular markers of cancer classification andoutcome prediction in a variety of tumour types.

RNA Isolation, Purification, and Amplification

General methods for mRNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al., Current Protocols of Molecular Biology, John Wiley and Sons(1997). Methods for RNA extraction from paraffin embedded tissues aredisclosed, for example, in Rupp and Locker, Lab Invest. 56: A67 (1987),and De Sandres et al., BioTechniques 18: 42044 (1995). In particular,RNA isolation can be performed using purification kit, buffer set, andprotease from commercial manufacturers, such as Qiagen, according to themanufacturer's instructions. For example, total RNA from cells inculture can be isolated using Qiagen RNeasy mini-columns. Othercommercially available RNA isolation kits include MasterPure CompleteDNA and RNA Purification Kit (EPICENTRE (D, Madison, Wis.), and ParaffinBlock RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samplescan be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumourcan be isolated, for example, by cesium chloride density gradientcentrifugation.

The steps of a representative protocol for profiling gene expressionusing fixed, paraffin-embedded tissues as the RNA source, including mRNAisolation, purification, primer extension and amplification are given invarious published journal articles (for example: T. E. Godfrey et al. J.Molec. Diagnostics 2: 84-91 (2000); K. Specht et al., Am. J. Pathol.158: 419-29 (2001)). Briefly, a representative process starts withcutting about 10 μm thick sections of paraffin-embedded tumour tissuesamples. The RNA is then extracted, and protein and DNA are removed.After analysis of the RNA concentration, RNA repair and/or amplificationsteps may be included, if necessary, and RNA is reverse transcribedusing gene specific promoters followed by RT-PCR. Finally, the data areanalyzed to identify the best treatment option(s) available to thepatient on the basis of the characteristic gene expression patternidentified in the tumour sample examined.

Immunohistochemistry and Proteomics

Immunohistochemistry methods are also suitable for detecting theexpression levels of the proliferation markers of the present invention.Thus, antibodies or antisera, preferably polyclonal antisera, and mostpreferably monoclonal antibodies specific for each marker, are used todetect expression. The antibodies can be detected by direct labeling ofthe antibodies themselves, for example, with radioactive labels,fluorescent labels, hapten labels such as, biotin, or an enzyme such ashorse radish peroxidase or alkaline phosphatase. Alternatively,unlabeled primary antibody is used in conjunction with a labeledsecondary antibody, comprising antisera, polyclonal antisera or amonoclonal antibody specific for the primary antibody.Immunohistochemistry protocols and kits are well known in the art andare commercially available.

Proteomics can be used to analyze the polypeptides present in a sample(e.g., tissue, organism, or cell culture) at a certain point of time. Inparticular, proteomic techniques can be used to assess the globalchanges of polypeptide expression in a sample (also referred to asexpression proteomics). Proteomic analysis typically includes: (1)separation of individual polypeptides in a sample by 2-D gelelectrophoresis (2-D PAGE); (2) identification of the individualpolypeptides recovered from the gel, e.g., by mass spectrometry orN-terminal sequencing, and (3) analysis of the data usingbioinformatics. Proteomics methods are valuable supplements to othermethods of gene expression profiling, and can be used, alone or incombination with other methods, to detect the products of theproliferation markers of the present invention.

Hybridization Methods Using Nucleic Acid Probes Selective for a Marker

These methods involve binding the nucleic acid probe to a support, andhybridizing under appropriate conditions with RNA or cDNA derived fromthe test sample (Sambrook, J., E Fritsch, E. and T Maniatis, MolecularCloning: A Laboratory Manual 3^(rd). Cold Spring Harbor LaboratoryPress: Cold Spring Harbor (2001)). These methods can be applied to BTMderived from a tumour tissue or fluid sample. The RNA or cDNApreparations are typically labeled with a fluorescent or radioactivemolecule to enable detection and quantification. In some applications,the hybridizing DNA can be tagged with a branched, fluorescently labeledstructure to enhance signal intensity (Nolte, F. S., Branched DNA signalamplification for direct quantitation of nucleic acid sequences inclinical specimens. Adv. Clin. Chem. 33, 201-35 (1998)). Unhybridizedlabel is removed by extensive washing in low salt solutions such as0.1×SSC, 0.5% SDS before quantifying the amount of hybridization byfluorescence detection or densitometry of gel images. The supports canbe solid, such as nylon or nitrocellulose membranes, or consist ofmicrospheres or beads that are hybridized when in liquid suspension. Toallow washing and purification, the beads may be magnetic (Haukanes, B-Iand Kvam, C., Application of magnetic beads in bioassays. Bio/Technology11, 60-63 (1993)) or fluorescently-labeled to enable flow cytometry (seefor example: Spiro, A., Lowe, M. and Brown, D., A Bead-Based Method forMultiplexed Identification and Quantitation of DNA Sequences Using FlowCytometry. Appl. Env. Micro. 66, 4258-4265 (2000)).

A variation of hybridization technology is the QuantiGene Plex® assay(Genospectra, Fremont) which combines a fluorescent bead support withbranched DNA signal amplification. Still another variation onhybridization technology is the Quantikine® mRNA assay (R&D Systems,Minn.). Methodology is as described in the manufacturer's instructions.Briefly the assay uses oligonucleotide hybridization probes conjugatedto Digoxigenin. Hybridization is detected using anti-Digoxigeninantibodies coupled to alkaline phosphatase in colorometric assays.

Additional methods are well known in the art and need not be describedfurther herein.

Enzyme-Linked Immunological Assays (ELISA)

Briefly, in sandwich ELISA assays, a polyclonal or monoclonal antibodyagainst the BTM is bound to a solid support (Crowther, J. R. The ELISAguidebook. Humana Press: New Jersey (2000); Harlow, E. and Lane, D.,Using antibodies: a laboratory manual. Cold Spring Harbor LaboratoryPress: Cold Spring. Harbor (1999)) or suspension beads. Other methodsare known in the art and need not be described herein further.Monoclonal antibodies can be hybridoma-derived or selected from phageantibody libraries (Hust M. and Dubel S., Phage display vectors for thein vitro generation of human antibody fragments. Methods Mol Biol.295:71-96 (2005)). Non-specific binding sites are blocked withnon-target protein preparations and detergents. The capture antibody isthen incubated with a preparation of urine or tissue containing the BTMantigen. The mixture is washed before the antibody/antigen complex isincubated with a second antibody that detects the target BTM. The secondantibody is typically conjugated to a fluorescent molecule or otherreporter molecule that can either be detected in an enzymatic reactionor with a third antibody conjugated to a reporter (Crowther, Id.).Alternatively, in direct ELISAs, the preparation containing the BTM canbe bound to the support or bead and the target antigen detected directlywith an antibody-reporter conjugate (Crowther, Id.).

Methods for producing monoclonal antibodies and polyclonal antisera arewell known in the art and need not be described herein further.

Immunodetection

The methods can also be used for immunodetection of marker familymembers in sera or plasma from bladder cancer patients taken before andafter surgery to remove the tumour, immunodetection of marker familymembers in patients with other cancers, including but not limited to,colorectal, pancreatic, ovarian, melanoma, liver, oesophageal, stomach,endometrial, and brain and immunodetection of marker family members inurine and stool from bladder cancer patients.

BTMs can also be detected in tissues or urine using other standardimmunodetection techniques such as immunoblotting or immunoprecipitation(Harlow, E. and Lane, D., Using antibodies: a laboratory manual. ColdSpring Harbor Laboratory Press: Cold Spring Harbor (1999)). Inimmunoblotting, protein preparations from tissue or fluid containing theBTM are electrophoresed through polyacrylamide gels under denaturing ornon-denaturing conditions. The proteins are then transferred to amembrane support such as nylon. The BTM is then reacted directly orindirectly with monoclonal or polyclonal antibodies as described forimmunohistochemistry. Alternatively, in some preparations, the proteinscan be spotted directly onto membranes without prior electrophoreticseparation. Signal can be quantified by densitometry.

In immunoprecipitation, a soluble preparation containing the BTM isincubated with a monoclonal or polyclonal antibody against the BTM. Thereaction is then incubated with inert beads made of agarose orpolyacrylamide with covalently attached protein A or protein G. Theprotein A or G beads specifically interact with the antibodies formingan immobilized complex of antibody-BTM-antigen bound to the bead.Following washing the bound BTM can be detected and quantified byimmunoblotting or ELISA.

Threshold Determination

For tests using down-regulated BTMs in either ratios or regressionanalyses, thresholds will be derived that will enable a sample to becalled either positive or negative for TCC. These thresholds will bedetermined by the analysis of cohorts of patients who are beinginvestigated for the presence of TCC. Thresholds may vary for differenttest applications; for example, thresholds for use of the test inpopulation screening will be determined using cohorts of patients whoare largely free of urological symptoms, and these thresholds may bedifferent to those used in tests for patients who are under surveillancefor TCC recurrence, or those being investigated for the presence ofurological symptoms such as hematuria. A threshold could be selected toprovide a practical level of test specificity in the required clinicalsetting; that is, a specificity that allows reasonable sensitivitywithout excessive numbers of patients receiving false positive results.This specificity may be within the range of 80-90%. An alternativemethod to obtain a test threshold is to plot sensitivity againstspecificity for different test thresholds (ROC curves) then select thepoint of inflexion of the curve.

As an alternative to single thresholds, the test may use test intervalswhich provide different degrees of likelihood of presence of disease andwhich have different clinical consequences associated with them. Forexample, a test may have three intervals; one associated with a high (eg90%) risk of the presence of TCC, a second associated with a low risk ofTCC and a third regarded as being suspicious of disease. The“suspicious” interval could be associated with a recommendation for arepeat test in a defined period of time.

Methods for Detecting Bladder Cancer Markers in Urine

In several embodiments, assays for BTM can be desirably carried out onurine samples. In general, methods for assaying for oligonucleotides,proteins and peptides in these fluids are known in the art. However, forpurposes of illustration, urine levels of a BTM can be quantified usinga sandwich-type enzyme-linked immunosorbent assay (ELISA). For plasma orserum assays, a 5 μL aliquot of a properly diluted sample or seriallydiluted standard BTM and 75 μL of peroxidase-conjugated anti-human BTMantibody are added to wells of a microtiter plate. After a 30-minuteincubation period at 30° C., the wells are washed with 0.05% Tween 20 inphosphate-buffered saline (PBS) to remove unbound antibody. Boundcomplexes of BTM and anti-BTM antibody are then incubated witho-phenylendiamine containing H₂O₂ for 15 minutes at 30° C. The reactionis stopped by adding 1 M H₂SO₄, and the absorbance at 492 nm is measuredwith a microtiter plate reader. It can be appreciated that anti-BTMantibodies can be monoclonal antibodies or polyclonal antisera.

Because many proteins are either (1) secreted by cells, (2) cleaved fromcell membranes, (3) lost from cells upon cell death or (4) containedwithin sloughed cells, it will be appreciated that BTMs may also bedetected in the urine. Additionally, diagnosis of bladder cancer can bedetermined by measuring either expression of BTMs in a sample, oraccumulation of BTMs in a sample. Prior art methods of diagnosis includecystoscopy, cytology and examination of cells extracted during theseprocedures. Such methods have relied upon identification of tumour cellsin the urine or in a brush sample of urothelium, or in other cases, inbiopsy specimens of the bladder wall. These methods suffer from severaltypes of errors, including sampling error, errors in identificationbetween observers, and the like.

Antibodies to Bladder Tumour Markers

In additional aspects, this invention includes manufacture of antibodiesagainst BTMs. Using methods described herein, novel BTMs can beidentified using microarray and/or qPCR methods. Once a putative markeris identified, it can be produced in sufficient amount to be suitablefor eliciting an immunological response. In some cases, a full-lengthBTM can be used, and in others, a peptide fragment of a BTM may besufficient as an immunogen. The immunogen can be injected into asuitable host (e.g., mouse, rabbit, etc) and if desired, an adjuvant,such as Freund's complete adjuvant, Freund's incomplete adjuvant can beinjected to increase the immune response. It can be appreciated thatmaking antibodies is routine in the immunological arts and need not bedescribed herein further. As a result, one can produce antibodiesagainst BTMs identified using methods described herein.

In yet further embodiments, antibodies can be made against the proteinor the protein core of the tumour markers identified herein or againstan oligonucleotide sequence unique to a BTM. Although certain proteinscan be glycosylated, variations in the pattern of glycosylation can, incertain circumstances, lead to mis-detection of forms of BTMs that lackusual glycosylation patterns. Thus, in certain aspects of thisinvention, BTM immunogens can include deglycosylated BTM ordeglycosylated BTM fragments. Deglycosylation can be accomplished usingone or more glycosidases known in the art. Alternatively, BTM cDNA canbe expressed in glycosylation-deficient cell lines, such as prokaryoticcell lines, including E. coli and the like.

Vectors can be made having BTM-encoding oligonucleotides therein. Manysuch vectors can be based on standard vectors known in the art. Vectorscan be used to transfect a variety of cell lines to produceBTM-producing cell lines, which can be used to produce desiredquantities of BTM for development of specific antibodies or otherreagents for detection of BTMs or for standardizing developed assays forBTMs.

Kits

Based on the discoveries of this invention, several types of test kitscan be envisioned and produced. First, kits can be made that have adetection device pre-loaded with a detection molecule (or “capturereagent”). In embodiments for detection of BTM mRNA, such devices cancomprise a substrate (e.g., glass, silicon, quartz, metal, etc) on whicholigonucleotides as capture reagents that hybridize with the mRNA to bedetected is bound. In some embodiments, direct detection of mRNA can beaccomplished by hybridizing mRNA (labeled with cy3, cy5, radiolabel orother label) to the oligonucleotides on the substrate. In otherembodiments, detection of mRNA can be accomplished by first makingcomplementary DNA (eDNA) to the desired mRNA. Then, labeled cDNA can behybridized to the oligonucleotides on the substrate and detected.

Antibodies can also be used in kits as capture reagents. In someembodiments, a substrate (e.g., a multiwell plate) can have a specificBTM capture reagent attached thereto. In some embodiments, a kit canhave a blocking reagent included. Blocking reagents can be used toreduce non-specific binding. For example, non-specific oligonucleotidebinding can be reduced using excess DNA from any convenient source thatdoes not contain BTM oligonucleotides, such as salmon sperm DNA.Non-specific antibody binding can be reduced using an excess of ablocking protein such as serum albumin. It can be appreciated thatnumerous methods for detecting oligonucleotides and proteins are knownin the art, and any strategy that can specifically detect BTM associatedmolecules can be used and be considered within the scope of thisinvention.

In addition to a substrate, a test kit can comprise capture reagents(such as probes), washing solutions (e.g., SSC, other salts, buffers,detergents and the like), as well as detection moieties (e.g., cy3, cy5,radiolabels, and the like). Kits can also include instructions for useand a package.

BTM ratios Used for Detection of Bladder Cancer I

In one series of embodiments, reagents for the testing the BTM LTBDH4 incombination with over-expressing BTMs can be incorporated into a kit forthe testing of unfractionated urine or urine cell sediments to detectbladder cancer. The urine samples could be collected from patients withdiagnosed bladder cancer who require monitoring for disease progressionor treatment response, individuals with urological symptoms includingmacroscopic or microscopic hematuria, or asymptomatic individuals. Forpatients or individuals being tested with a kit that measures the BTMsin unfractionated urine, approximately 2 mls of urine can be taken fortesting. For tests on the urine pellet, >10 mls of urine can becollected.

A suitable kit includes: (i) instructions for use and resultinterpretation, (ii) software for interpretation of multiple geneanalyses, including any regression analysis classifier or formula (iii)reagents for the stabilization and purification of RNA fromunfractionated urine or urine pellets, (iv) reagents for the synthesisof cDNA including dNTPs and reverse transcriptase, and (v) reagents forthe quantification of the BTM cDNA. In one form, these reagents would beused for quantitative PCR and would include specific exon-spanningoligonucleotide primers, a third oligonucleotide labeled with a probefor detection, Taq polymerase and the other buffers, salts and dNTPsrequired for PCR. The kit can also use other methods for detection ofthe transcripts such as direct hybridization of the BTM RNA with labeledprobes or branched DNA technology; and (vi) oligonucleotides and probefor the detection of transcripts from a highly transcribed gene, such asβ-actin: to serve as a quality control measure.

Evaluation of Progression of Bladder Cancer Using BTM ratios

To evaluate the progression of bladder tumours, samples of tissue areobtained by biopsy of bladder wall or samples of urine are collectedover time from a patient having bladder cancer. Evaluation of the ratioof BTMs or combinations thereof are made for samples taken at differenttimes. BTM ratios within a specified range are indicative of progressionof bladder cancer.

Evaluation of Therapy of Bladder Cancer Using BTM Ratios

To evaluate the efficacy of therapy for bladder tumours, samples oftissue and/or urine are obtained before treatment is initiated. Thebaseline levels of one or more BTMs are determined, as are ratios ofvarious BTMs with respect to each other. Treatment is initiated, and caninclude any therapy known in the art, including surgery, radiationtherapy or chemotherapy as appropriate to the type and stage of thedisease. During the course of therapy, samples of tissue and/or urineare collected and analyzed for the presence and amount of BTMs. Ratiosof various BTMs are determined and results are compared to: (1) thepatient's baseline levels before treatment or (2) normal values obtainedfrom a population of individuals not having bladder cancer.

Use of BTM Ratios to Monitor the Progression of TCC Therapies

In addition to the rapid diagnosis and early detection of TCC; BTMmarker ratios detected in either tissue, serum or urine can be used tomonitor a patient's response to therapy. In these applications, urineand/or serum samples can be taken at intervals following the initiationof systemic, intravesicular or intravascular chemotherapy, radiotherapyor immunotherapy. A change in marker ratio can indicate a reduction intumour size, indicative of effective treatment. The rate of change canbe used to predict the optimum therapeutic dose for each patient ortreatment.

Use of BTM Regression Analyses

In addition to the BTM ratios, regression or classification analysesthat include high and low expressing BTM family members can be used forthe applications described above.

Markers evaluated are selected from known human genes. The genesevaluated are indicated in FIGS. 3 and 4. Included in FIGS. 3 and 4 arethe name of the gene, the HUGO identifier, MWG oligo number, NCBI mRNAreference sequence number and the protein reference number. The fulllength sequences can be found at http://www.ncbi.nlm.nih.gov/entrez/.

EXAMPLES

The examples described herein are for purposes of illustratingembodiments of the invention and are not intended to limit the scope ofthe invention. Other embodiments, methods and types of analyses arewithin the scope of persons of ordinary skill in the moleculardiagnostic arts and need not be described in detail hereon. Otherembodiments within the scope of the art that are based on the teachingsherein are considered to be part of this invention.

Methods

Tumour Collection

Bladder tumour samples and non-malignant urothelium samples werecollected from surgical specimens resected at Kyoto University Hospital,Japan.

Urine Collection

Urine samples from non-malignant controls and bladder cancer patientswere obtained from Kyoto University Hospital, Japan. Healthy controlsamples were obtained from Japanese volunteers (FIG. 1).

RNA Extraction

Tumour tissues were homogenized in a TriReagent: water (3:1) mix, thenchloroform extracted. Total RNA was then purified from the aqueous phaseusing the RNeasy™ procedure (Qiagen). RNA was also extracted from 16cancer cell lines and pooled to serve as a reference RNA.

RNA was extracted from urine by mixing the urine sample with an equalvolume of lysis buffer (5.64 M guanidine-HCl, 0.5% sarkosyl, 50 mMsodium acetate (pH 6.5) and 1 mM β-mercaptoethanol; pH adjusted to 7.0with 1.5 M Hepes pH 8). Due to the low amounts of RNA in urine, 7.5 ugsof total bacterial RNA was added to the urine/lysis buffer mix to act asa carrier. Total RNA was then extracted using Trizol and the RNeasy™procedure. RNA preparations were further purified prior to cDNAsynthesis using the Qiagen QIAquick™ PCR purification kit.

RNA was extracted from the blood of three healthy volunteers byperforming a Trizol/RNeasy™ extraction on cells enriched from wholeblood using sedimentation in 3.6% dextran.

Microarray Slide Preparation

Epoxy coated glass slides (MWG Biotech) were printed with ˜30,000 50 meroligonucleotides (MWG Biotech) using a Gene Machines microarrayingrobot, according to the manufacturer's protocol.

RNA Labeling and Hybridization

cDNA was transcribed from 5 μg total RNA using Superscript II™ reversetranscriptase (Invitrogen) in reactions containing5-(3-aminoallyl)-2′deoxyuridine-5′-triphosphate. The reaction was thende-ionised in a Microcon column before being incubated with Cy3 or Cy5in bicarbonate buffer for 1 hour at room temperature. Unincorporateddyes were removed using a Qiaquick column (Qiagen) and the sampleconcentrated to 15 μl in a SpeedVac. Cy3 and Cy5 labeled cDNAs were thenmixed with Ambion ULTRAhyb™ buffer, denatured at 100° C. for 2 min andhybridized to the microarray slides in hybridisation chambers at 42° C.for 16 hours. The slides were then washed and scanned twice in an Axon4000A™ scanner at two power settings.

Microarray Analysis of Cancer Marker Genes

RNA from 53 bladder tumours and 20 non-malignant (“normal”) bladdertissue samples were labeled with Cy5 and hybridized in duplicate ortriplicate with Cy3 labeled reference RNA. After normalization, thechange in expression in each of 29,718 genes was then estimated by foldchange and statistical probability.

Normalisation Procedure

Median fluorescence intensities detected by Genepix™ software werecorrected by subtraction of the local background intensities. Spots witha background corrected intensity of less than zero were excluded. Tofacilitate normalization, intensity ratios and overall spot intensitieswere log-transformed. The logged intensity ratios were corrected for dyeand spatial bias using local regression implemented in the LOCFIT™package. Logged intensity ratios were regressed simultaneously withrespect to overall spot intensity and location. The residuals of thelocal regression provided the corrected logged fold changes. For qualitycontrol, ratios of each normalized microarray were plotted in respect tospot intensity and localization. The plots were subsequently visuallyinspected for any remaining artifacts. Additionally, an ANOVA model wasapplied for the detection of pin-tip bias. All results and parameters ofthe normalization were inserted into a Postgres-database for statisticalanalysis.

Statistical Analysis

To improve the comparison of measured fold changes between arrays, log 2(ratios) were scaled to have the same overall standard deviation perarray. This standardization reduced the average within-tissue classvariability. A rank-test based on fold changes was then used to improvethe noise robustness. This test consists of two steps: (i) calculationof the rank of fold change (Rfc) within arrays and ii) subtraction ofthe median (Rfc) for normal tissue from the median (Rfc) for tumourtissue. The difference of both median ranks defines the score of thefold change rank. Three additional statistical tests were also performedon standardized data: 1) Two sample student's t-test, 2) the Wilcoxontest and 3) Statistical Analysis of Microarrays (SAM). The mostsignificantly down-regulated genes determined by each of the statisticalmethods (rank fold change, t-test, Wilcoxon test, and SAM) were given arank score for each test. All rank scores were then added into onesummated rank score.

cDNA Synthesis from Urine RNA

Total urine RNA was annealed to gene-specific primers for each of thebladder tumour markers by incubating at 70° C. then cooling on ice for 2mins in 50 ul reactions containing forward primers at 0.01 μg/μl. EachcDNA reaction contained annealed RNA and 4 μl of 5× Superscript IIreverse transcriptase buffer (Invitrogen, USA), 2 μl of 0.1M DTT(Invitrogen, USA), 0.5 μl of RNase out (40 U/μL), (Invitrogen, USA), 4μl of 10 mM dNTP (Invitrogen, USA) and 0.5 μl of Superscript II reversetranscriptase (200 U/μl), (Invitrogen, USA) in a final volume of 20 μl.Reactions were incubated at 42° C. for 1 hour, 10 minutes at 70° C. and1 minute at 80° C. Reactions were cleaned prior to qPCR with QiagenQIAquick PCR purification columns (Qiagen, Victoria, Australia) andstored at −80° C.

Quantitative Real-Time PCR

Real-time or quantitative PCR (qPCR) is used for absolute or relativequantitation of PCR template copy number. Taqman™ probe and primer setswere designed using Primer Express V 2.0™ (Applied Biosystems). Wherepossible, all potential splice variants were included in the resultingamplicon, with amplicon preference given to regions covered by theMWG-Biotech-derived microarray oligonucleotide. Primer and probesequences are shown in FIG. 2. Alternatively, if the target gene wasrepresented by an Assay-on-Demand™ expression assay (Applied Biosystems)covering the desired amplicons, these were used. In the in-housedesigned assays, primer concentration was titrated using a SYBR greenlabeling protocol and cDNA made from the reference RNA. Amplificationwas carried out on an ABI Prism™ 7000 sequence detection system understandard cycling conditions. When single amplification products wereobserved in the dissociation curves, standard curves were generated overa 625 fold concentration range using optimal primer concentrations and5′FAM-3′TAMRA phosphate Taqman™ probe (Proligo) at a final concentrationof 250 nM. Assays giving standard curves with regression coefficientsover 0.98 were used in subsequent analyses.

Assays were performed in 96 well plates. Each plate contained areference cDNA standard curve, over a 625-fold concentration range. Forthe urine qPCR, total RNA extracted from ˜0.5 mls unfractionated urinewas used in each reaction. The ΔCt (target gene Ct-mean reference cDNACt) was calculated for each marker, and used in subsequent ratios,regression or classification analysis.

Expression of Markers in Blood

The expression of the markers shown in FIGS. 3 and 4 in whole blood wasdetermined in silico. Microarray probes were linked to UniGene clustersvia the GenBank accession numbers of their target mRNAs, and the tissueexpression profile from UniGene used to determine the number ofexpressed sequence tags (ESTs) in blood libraries. Only genes with 0 or1 expressed sequence tags (EST) are shown in FIG. 4. To confirm the lowexpression of LTB4DH in whole blood, RT-qPCR was carried out on totalRNA extracted from whole blood using the primers and probes shown inFIG. 2. No significant expression was observed (results not shown).

Identification of Down Regulated Bladder Cancer Markers

To identify down-regulated markers of bladder cancer, we performedmicroarray studies on RNA from 53 bladder tumours and 20 non-malignantbladder tissue samples using 30,000 oligonucleotide chips. FIG. 3 showsthe statistical analysis of microarray data for 300 genes that showsignificant downregulation in bladder cancer tissue compared tonon-malignant tissue. FIG. 3 includes the HUGO gene name and symbol, theprotein reference sequence number, the NCBI mRNA reference sequencenumber, the MWG Biotech probe oligonucleotide number, the median foldchange in gene expression between tumour and non-malignant tissue, theresults of an original unadjusted Student's t-test, the results of the2-sample Wilcoxon test, the results of the SAM test, and the summatedrank score.

Identification of Preferred Under-Expressed Bladder Tumour Markers forUse in Urine Tests for Bladder Cancer

Because urinary hematuria is a common co-occurrence with bladder cancer,it is an advantage that bladder cancer markers are not significantlyelevated in whole blood. In addition, because the downregulated markersare being used in ratios, regression or classification analysis, it isan advantage that they be present in sufficiently high copy numbers inboth tumour cells and non-malignant bladder cells to enable reliabledetection in urine. To identify suitable markers, we screened the genesin FIG. 3 for a subset that had little or no representation in blood ESTlibraries, and had higher than median expression in non-malignanttissue. Median expression was estimated by ranking the 30,000oligonucleotides on the array by their median intensity in the samplesanalysed in the microarray study. Markers that met the criteria areshown in FIG. 4. FIG. 4 includes the HUGO gene name and symbol, theprotein reference sequence number, the NCBI mRNA reference sequencenumber, the median fold change, the rank score, the median rank ofmicroarray spot intensity in tumour tissue and non-malignant tissue, andthe number of ESTs present in blood EST libraries.

The down regulation observed in the array data was validated by qPCR forthree genes shown in FIG. 4, LTB4DH, BAG1 and FLJ21511. These genes weretested on total RNA from 10 tumour samples and 10 non-malignant samples.LTB4DH, BAG1 and F1121511 showed an average downregulation in bladdertumours compared to bladder non-malignant tissue of 2.5 fold, 1.4 foldand 6.1 fold, respectively, in these samples.

qPCR Analysis of Urine using LTB4DH

Urine from TCC patients and controls with non-malignant urologicalconditions was collected by either voiding or catheterisation. Total RNAwas extracted from the voided urine of 42 controls and the voided orcatheterised urine of 37 TCC patients and used in quantitative RT-PCRusing primers and probes for LTB4DH and three over-expressed markers,IGFBP5, MDK and HoxA13. The ΔCt ratios were determined forIGFBP5/LTB4DH, MDK/LTB4DH and HoxA13/LTB4DH. This data is illustrated bythe box plots in FIG. 5, which show a clear difference in the spread ofdata between the urine samples from controls and TCC patients for eachof the three tests. The most accurate test was IGFBP5/LTB4DH whichdemonstrated sensitivity and specificity of 87% and 88% in this samplecohort, respectively (FIG. 6a ). To illustrate the correspondencebetween sensitivity and specificity for each of these tests, ROC curvesare shown in FIG. 7. The areas under the curve for IGFBP5/LTB4DH,MDK/LTB4DH and HoxA13/LTB4DH are 0.9223, 0.9199, and 0.7497,respectively. These areas, which measure test accuracy, indicate thatall three ratios with LTB4DH are useful tests, in particularIGFBP5/LTB4DH and MDK/LTB4DH.

To increase the sensitivity and specificity of TCC detection,combinations of two tests were used. The optimal sensitivities andspecificities of these test combinations are shown in FIG. 6b . FIG.8a-f shows the separation of data in 2 dimensional space for each of thethree tests using LTB4DH and BAG1. This data shows that combinations oftwo or more tests that include either of the downregulated BTMs LTB4DHor BAG1, are able to achieve sensitivities and specificities of over90%. Moreover, because these, tests are measuring simple gene expressionsignatures and not absolute levels of markers, they will be robust tovariations in urine concentration.

To demonstrate the robustness of tests involving ratios with LTB4DH tourine concentration, the levels of IGFBP5 alone (ΔCt) and IGFBP5/LTB4DHwere plotted as a function of urine concentration (FIG. 9a-b ) andtrendlines fitted to the data. It can be seen that for both urinesamples from non-malignant controls and patients with TCC, there is adecrease in the IGFBP5 ΔCt with increasing urine concentration that isabsent in the IGFBP5/LTB4DH ratio. The effect is most pronounced withthe non-malignant samples because of the absence of other influencessuch as tumour size and tumour heterogeneity in the expression of IGFBP5and LTB4DH.

In some instances, when single markers are used in bladder cancerassays, the method of urine sample collection can affect the amount ofmarker detected due to variations in the number of exfoliated bladdercells collected. This bias could lead to false positive or falsenegative results in a small proportion of samples. The use of ratiosincluding LTB4DH or other low-expressing genes should provide a methodto compensate for different methods. To test this hypothesis, samplescollected from TCC patients by either simple voiding (nine samples) orcatheterisation (28 samples) were tested for the presence of TCC markersand LTB4DH. Analysis of the TCC markers alone showed that the voidedsamples were more heavily represented at the lower end of the range ofdata (higher Ct), consistent with a lower average number of exfoliatedcells in these samples compared to the catheterised samples. This isillustrated in the self-self scatter plots for IGFBP5, MDK and HoxA13 inFIG. 10a-c . In contrast, when ratios between these markers and LTB4DHwere calculated, the voided and catheterised samples were spread oversimilar ranges of Ct ratios (FIG. 10d-f ), illustrating that thecalculation of gene expression signatures between high expressingmarkers and low expressing markers such as LTB4DH compensate forvariations in marker levels introduced by different urine samplingmethodologies.

Urine samples from patients with low grade tumours are often borderlinein their accumulation of BTMs due to the presence of only small numbersof exfoliated cells in these samples. These samples are therefore athigh risk of being incorrectly classified due to variations in samplingmethod or urine concentration.

The utility of gene expression ratios that incorporate down-regulatedgenes for the detection of TCC is therefore likely to be pronounced whenapplied to the detection of low grade TCC. To demonstrate this effect, acohort of voided 43 urine samples from patients with low grade TCC and123 controls were tested with the markers IGFBP5, HoxA13 and LTB4DH. Theclinical characteristics of the cohort are summarised in FIG. 13. TheqPCR data for IGFBP5 and HoxA13 were analysed alone and in ratios withLTB4DH using the area under the ROC curve as a measure of test accuracy(STATA statistics package). The results are summarized in FIG. 14. Usingthe IGFBP5 marker, LTB4DH increased the accuracy of detection of lowgrade (grade 1-2) stage Ta TCCs by 9% and low grade TCCs of any stage by8%. The accuracy of HoXA13 testing of low grade stage Ta TCCs wasincreased by 3%.

Linear Discriminate Analysis of qPCR Data using LTB4DH

Linear discriminate analysis (LDA) is a statistical technique (Fisher R.A. “The Use of Multiple Measurements in Taxonomic Problems”, Annals ofEugenics 7 179 (1936)) in which a linear combination of variables isgenerated, such that there is maximal separation between two or moregroups. This linear combination of variables is termed the “lineardiscriminant”, which is a linear function of the input variables thatmaximally separates the classes of the data set. The ability of LDA (orany other classification technique) to characterise a particulardataset, such as qPCR data, can be tested using cross-validation. Inthis method, part of the dataset is used to generate a classifier, andpart of the dataset is used to measure the effectiveness of thatclassifier. The partitioning of the dataset into training and testingsets can be repeated multiple times (each time generating a newclassifier). In k-fold cross-validation, the dataset is split k-wise,and each subset is used as the testing set in one of k rounds oftraining and validation. This can be extended to leave-one-outcross-validation (LOOCV) where each sample is classified according to aclassifier generated from the remaining samples in the dataset (“leavingone out”; leaving out the sample which is being tested).

LDA and LOOCV were used to illustrate the utility of the downregulatedBTM, LTB4DH, in improving the diagnosis of TCC. qPCR was first carriedout on the cohort of control and TCC urine samples described in FIG. 13which were supplemented with an additional 30 grade 3 tumours (5>stage1, 13=stage 1, 4=Tis, and 8=Ta). Combinations of the six genes LTB4DH,MDK, 1GFBP5, HOXA13, TOP2a and CDC2 were tested for classifierperformance, as judged by LOOCV. The posterior probability (that thesample “left out” was a TCC sample) was used to generate ROC curvesusing the ROCR package of the R statistical programming environment. Thesensitivity of the classifier for a given specificity was obtained byreference to the appropriate ROC curve.

The sensitivity of detection of TCC using combinations of upregulatedBTMs with and without LTB4DH was determined at a specificity of 85%. Theresults of this analysis are shown in FIG. 15. It can be seen that theaddition of LTB4DH to assays including combinations of the upregulatedBTMs MDK, IGFBP5, Top2a, cdc2 and HoxA13 increased the overallsensitivity by 1-2% and the sensitivity of detection of Stage Tatumours, grade 1-2 tumours and grade 3 tumours by up to 3%.

Wherein in the foregoing description reference has been made to integersor components having known equivalents, such equivalents are hereinincorporated as if individually set fourth.

Although the invention has been described by way of example and withreference to possible embodiments thereof, it is to be appreciated thatimprovements and/or modifications may be made without departing from thescope or the spirit thereof.

INDUSTRIAL APPLICABILITY

Methods for detecting BTM family members include detection of nucleicacids, proteins and peptides using microarray and/or real time PCRmethods. The compositions and methods of this invention are useful indiagnosis of disease, evaluating efficacy of therapy, and for producingreagents and test kits suitable for measuring expression of BTM familymembers.

The invention claimed is:
 1. A testing device, comprising: a substratedivided into a plurality of locations thereon wherein, i) one of saidplurality of locations has an oligonucleotide having the sequence of SEQID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 that bind to an oligonucleotideof IGFBP5; ii) another one of said locations having an oligonucleotidethat binds to an oligonucleotide of LTB4DH; iii) another one of saidlocations having an oligonucleotide that binds to an oligonucleotide ofHOXA13; iv) another one of said locations having an oligonucleotide thatbinds to an oligonucleotide of MDK; v) another of said locations havingan oligonucleotide that binds to an oligonucleotide of CDC2; and vi)another one of said locations having an oligonucleotide that binds to anoligonucleotide of BAG 1 wherein each of the oligonucleotides areimmobilized on the substrate.
 2. A method for determining the presenceof Transitional Cell Carcinoma (TCC) of the bladder in a subject,comprising: (a) providing a device of claim 1; (b) providing a sample ofurine from the subject; (c) providing a set of samples of urine from aplurality of subjects not having TCC; (d) for each of steps (b) and (c),(i) applying mRNA or cDNA made from mRNA obtained from said sampleobtained in steps (b) to said device of step (a); (ii) applying mRNA orcDNA made from mRNA obtained from each of said set of subjects in step(c); (e) applying a plurality of labeled oligonucleotides each of whichbinds to one of the genetic markers on said substrates; (f) washing saidsubstrates under stringent conditions; (g) quantifying the amount ofeach of said labeled oligonucleotides of step (e); (h) normalizing saidamounts of each of said labeled oligonucleotides of step (g) therebyproviding a measures of expression of each of said genetic markers insaid sample and said group of said subjects in step (d)(ii); (i) forsaid sample and said plurality of subjects not having TCC, calculatingthe ratios of expression of IGFBP5/LTB4DH, or the ratios of expressionof HOXA13/LTB4DH, or the ratios of expression of MDK/LTB4DH; (i) if oneor more of the ratios of said sample obtained in step (i) are lower thanthe ratios of said genetic markers for said plurality of subjects nothaving TCC, said patient has TCC.
 3. The device of claim 1, wherein saidoligonucleotide that binds to LTB4DH comprises SEQ ID NO: 4, or SEQ IDNO: 5, or SEQ ID NO:
 6. 4. The device of claim 1, wherein saidoligonucleotide that binds to CDC2 comprises SEQ ID NO: 7, or SEQ ID NO:8.
 5. The device of claim 1, wherein said oligonucleotide that binds toIGFBP5 comprises SEQ ID NO:
 1. 6. The device of claim 1, wherein saidoligonucleotide that binds to IGFBP5 comprises SEQ ID NO:
 2. 7. Thedevice of claim 1, wherein said oligonucleotide that binds to IGFBP5comprises SEQ ID NO:
 3. 8. The device of claim 1, wherein saidoligonucleotide that binds to LTB4DH comprises SEQ ID NO:
 4. 9. Thedevice of claim 1, wherein said oligonucleotide that binds to LTB4DHcomprises SEQ ID NO:
 5. 10. The device of claim 1, wherein saidoligonucleotide that binds to LTB4DH comprises SEQ ID NO:
 6. 11. Thedevice of claim 1, wherein said oligonucleotide that binds to CDC2comprises SEQ ID NO:
 7. 12. The device of claim 1, wherein saidoligonucleotide that binds to CDC2 comprises SEQ ID NO: 8.